Summary of Work: Research during the past year focused on defining the molecular mechanisms underlying the development of the mammalian olfactory system. Particular emphasis was placed on the identification of molecules which direct the establishment of the precise patterns of synaptic connectivity observed between the peripheral sensory neurons and their target neurons in the olfactory bulb. To identify molecules involved in this process, RT-PCR experiments were performed with RNA derived from neonatal olfactory epithelium and bulb tissues. Degenerate PCR primers were designed to amplify members of either the netrin, semaphorin, eph receptor-type kinase or LERK gene families. These gene families were selected for study based on their involvement in axonal guidance and/or target recognition in other systems. Using this approach, members of each of these gene families were isolated and, subsequently, cloned and sequenced. In situ hybridization studies indicated that several of these genes are highly expressed in the olfactory system. Of particular interest was the finding that some of these genes are expressed early in development during the time when synaptogenesis between olfactory sensory neurons and the olfactory bulb mitral cells is occurring. Furthermore, in some cases genes belonging to the same family were differentially expressed by different cell types; both mitral cell specific and olfactory sensory neuron specific expression patterns were observed. A second focus of research was the molecular characterization of odorant receptors. In order to examine the functional properties of odorant receptors including ligand specificity, G-protein coupling and selectivity, and ligand-dependent modifications, we are currently developing a heterologous cell expression system. Mammalian expression vectors containing the complete coding regions of three odorant receptors were constructed, and stable transfected cell lines were established. Cell lines producing high levels of odorant receptor mRNA are currently being evaluated for the production of functional odorant receptors.